The ultimate object of a drug resistance test in chemotherapy is to evaluate the effectiveness of drugs against causative microorganisms (also referred to hereinafter as pathogen) causing of infections for achieving appropriate drug administration.
On the other hand, there are cases where the pathogen has been rendered drug-resistant because of use of a large amount of anti-infection agents for a long period of time.
In this case, for the success of treatment of infections it is very important to ascertain the properties of the pathogen of interest, particularly with respect to drug sensitivity or drug resistance.
However, conventional drug sensitivity tests such as the disk methods micro-liquid dilution method (liquid medium dilution method) and agar plate dilution method (which are collectively referred to hereinafter as the conventional method) are disadvantaged in that because at least 16- to 20-hour incubation is required, evaluation of the results is obtained at the earliest the day after the examination is initiated, although highly reliable evaluation results can be obtained.
Under these circumstances, there is a demand for the advent of a method of testing microbial drug resistance that can be evaluated more rapidly.
Conventionally, a method of evaluating drug resistance by culturing a microorganism in a drug-containing medium for 3 to 5 hours and comparing the amount of ATP in the microorganism and the amount of ATP in the microorganism separately cultured in a medium not containing the drug is known as a method of testing microbial drug resistance that can be evaluated more rapidly (referred to hereinafter as the method of testing microbial drug sensitivity using the conventional ATP method) (Japanese Patent Appln. LOP Publication No. 370100/92).
This prior art method uses a luciferin-luciferase system luminescent reagent for the measurement of ATP levels. The following advantages are realized for this method: the detection limit of microorganisms is as high as 10.sup.3 CFU/ml which is very sensitive; the proliferation and deproliferation of microorganisms can be observed just after inoculation in the case of a drug sensitivity test where the amount of microorganisms inoculated into the medium is 10.sup.5 CFU/ml; and the culture time can be drastically reduced as compared with the conventional method (H. Hojer, L. Nilsson, S. Aosehn and A. Thore, Scand. J. Infect. Dis., Suppl., 9, 58-61, 1976 and A. Thore, L. Nilsson, E Hojer, S. Ansehn and L. Brote, Acta Path. Microbiol. Scand. Sect. B, 85, 161-166, 1977).
However, the results in this method of testing microbial drug sensitivity using the conventional ATP method often give contrary results i.e. false-resistant results when Gram-negative bacilli such as Pseudomonas aeruginosa etc. are examined for their resistance to .beta.-lactam antibiotics (Vellend, H., S. A. Tuttle, M. Barza, L. Weinstein, G. L. Picciolo and E. W. Chappelle, NASA Technical Notes 1974).
That is, the method of testing drug sensitivity using the conventional ATP method has a major drawback of giving false evaluation based on false-resistance of microorganisms to drugs, thus undermining the reliability of evaluation results.
As described in more detail below, it has been believed that the false-resistance give by the method of testing drug sensitivity using the conventional ATP method is caused by the fact that the microorganisms of interest, even while undergoing morphological transformation into, for example, spheroplast and filament forms by the influence of the drug, remain alive.
That is, this false-resistance is observed particularly frequently with .beta.-lactam antibiotics which inhibit the synthesis of cell walls in a microorganism, and if the microorganism is cultured in the presence of these antibiotics, the microorganism will survive initially while undergoing morphological transformation into spheroplast or filament forms, for example, but if culturing is further continued, the microorganism will finally lyse and perish owing to the difference in osmotic pressure in the surroundings. Accordingly, the microorganism while undergoing morphological transformation, is alive for 3 to 5 hours, which is a period of time required by the method to test for microbial drug resistance using the conventional ATP method, so in this case the result will be "resistant", whereas the microorganism perishes after 16 to 20 hours, which is a period of time required by the conventional method, so in this case the result will be "sensitive". Hence, false-resistance will be given by the method of testing microbial drug resistance using the conventional ATP method.
As countermeasures against this false-resistance evaluation by the method of testing microbial drug sensitivity using the conventional ATP method, there are known methods in which microorganisms morphologically transformed into forms of filament, spheroplast etc., by the influence of a drug are selectively lysed by diluting a medium or by using a medium of low osmotic pressure (P. F. Wheat, J. G. M. Hastings and R. C. Spencer, J. Med. Microbiol., 25, 95-99, 1988 and E. G. Hornsten, L. E. Nilsson, H. Elwing, and I. Lundstrom, Diagn. Microbiol. Infect. Dis., 12, 171-175, 1989).
However, these attempts are effective where the target is a specific microorganism, but these are not suitable for achieving the present object of the drug sensitivity test directed to a wide spectrum of microorganisms.
A method of measuring minimum inhibitory concentration using the conventional ATP method also has a major drawback of giving false evaluation based similarly on the result of false-resistance, thus undermining the reliability of evaluation results.
In addition, similar disadvantages exist in a method of measuring minimum bactericidal concentration (MBC), that is, the minimum amount of a drug at which the growth inhibitory effect of the drug on a microorganism acts in a bactericidal manners as well as in a method of measuring post antibiotic effect (PAE), that is, the effect of a drug which after exposure of a microorganism to the drug for a few hours, that lasts even in the absence of the drug.
Measurement of MBC is carried out by culturing a microorganism overnight in a drug-containing medium and then culturing it again in a drug-free medium to determine the minimum concentration of the drug at which the re-growth of the microorganism does not occur.
Measurement of PAE is carried out by culturing a microorganism for a few hours (about 2 hours) in a drug-containing medium, then diluting the culture 1000 times with a drug-free medium, and culturing the microorganism again, and determining the time at which the re-growth of the microorganism occurs.